The present invention relates generally to compounds and methods for the selective destruction of cells in a heterogenous cell population. The compounds feature, in pertinent part, a genotoxic agent that damages cellular DNA.
Frequently, a need arises in biological investigations and clinical or veterinary practice for selectively killing a subpopulation of cells in a heterogenous cell population. For example, to attain a strain or culture of cells having desirable characteristics, available in vitro techniques can be applied for selectively killing a subpopulation of cells in a heterogenous cell population that comprises cells that possess a desired characteristic. In this manner, cells that have undesirable characteristics can be eliminated from the population. Hybridoma cell lines producing desired monoclonal antibodies and stable genetic transfectant cell lines expressing the products of heterologous cloned genes are customarily established in this manner. See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. 1989). A mixed population of cells comprising the desired hybridoma or transfectant is maintained in culture for a period of time in the presence of one or more genotoxic drugs, such as aminopterin or methotrexate. The desired cells are resistant to the genotoxic effects of the drug employed. In contrast, other cells in the population are susceptible to the drug and fail to survive. These techniques rest on the creation of cells having a defined phenotype that confers resistance to a particular, preselected genotoxic drug. Thus, although significant advances in biology and biotechnology have been achieved through the use of these techniques, limits remain to their flexibility.
Another general context in which practitioners desire to kill cells selectively involves heterogenous cell populations comprising cells of two or more phylogenetically different species of organisms. Here, it may be desirable to destroy selectively the cells of one species while preserving viability of another. In this manner, a desired species can be enriched in the population or an offensive species, such as an infectious agent, can be removed. Here again, the desired objective is often accomplished by treating the cell population with a drug, such as an antibiotic, antiviral, antifungal or antiparasitic drug, to which the undesired species is susceptible. Cells of the undesired species succumb to the effects of the drug and die. Conversely, the desired species (e.g., a human or other host animal) must have the capacity to resist the chosen drug. Although a wide choice of drugs useful for such purposes has historically been available, recent reports have documented the appearance of drug resistance in undesirable species. For example, resistant strains of the organisms responsible for septic wound infections, hospital-acquired infections, tuberculosis, malaria, dysentery and a host of other contagious diseases have arisen in recent years. Harrison""s Principles of Internal Medicine, Part 5 Infectious Diseases, Ch. 78, 79, and 83-88 (12th ed. 1991). The emergence of such strains greatly complicates the treatment of infection, and limits choices available to the practitioner.
The need to manage or alleviate cancer provides yet another general setting in which practitioners require means for selectively killing cells in a heterogenous cell population. Here, the population comprises normal and neoplastic (malignant or transformed) cells in an individual""s tissues. Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual""s body thus proliferate, forming a tumor (also referred to as a neoplasm). When a neoplasm is found, the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment. Currently, three major approaches are followed for the clinical management of cancer in humans and other animals. Surgical resection of solid tumors, malignant nodules and or entire organs may be appropriate for certain types of neoplasia. For other types, e.g., those manifested as soluble (ascites) tumors, hematopoeitic malignancies such as leukemia, or where metastasis of a primary tumor to another site in the body is suspected, radiation or chemotherapy may be appropriate. Either of these techniques is also commonly used as an adjunct to surgery. Harrison""s Principles of Internal Medicine, Part 11 Hematology and Oncology, Ch. 296, 297 and 300-308 (12th ed. 1989).
Chemotherapy is based on the use of drugs that are selectively toxic to cancer cells. Id. at Ch. 301. Several general classes of chemotherapeutic drugs have been developed, including drugs that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes. These are generally referred to as antimetabolite drugs. Treatment regimes typically attempt to ensure inactivation of a particular pathway in cancer cell metabolism by coadministering two or more suitable antimetabolite drugs. Other classes of chemotherapeutic drugs inflict damage on cellular DNA. Drugs of these classes are generally referred to as genotoxic. The repair of damage to cellular DNA is an important biological process carried out by a cell""s enzymatic DNA repair machinery. Unrepaired lesions in a cell""s genome can impede DNA replication or impair the replication fidelity of newly synthesized DNA. Thus, genotoxic drugs are generally considered more toxic to actively dividing cells that engage in DNA synthesis than to quiescent, nondividing cells. In many body tissues, normal cells are quiescent and divide infrequently. Thus, greater time between rounds of cell division is afforded for the repair of damage to cellular DNA in normal cells. In this manner, practitioners can achieve some selectivity for the killing of cancer cells. Many treatment regimes reflect attempts to improve selectivity for cancer cells by coadministering chemotherapeutic drugs belonging to two or more of these general classes.
In some tissues, however, normal cells divide continuously. Thus, skin, hair follicles, buccal mucosa and other tissues of the gut lining, sperm and blood-forming tissues of the bone marrow remain vulnerable to the action of genotoxic drugs. These and other classes of chemotherapeutic drugs can also cause severe adverse side effects in drug-sensitive organs, such as the liver and kidneys. These and other adverse side effects seriously constrain the dosage levels and lengths of treatment regimens that can be prescribed for individuals in need of cancer chemotherapy. Id. at Ch. 301. See also Loehrer and Einhorn (1984), 100 Ann. Int. Med. 704-714 and Jones et al. (1985), 52 Lab. Invest. 363-374. Such constraints can predjudice the effectiveness of clinical treatment. For example, the drug or drug combination administered must contact and affect cancer cells at times appropriate to impair cell survival. Genotoxic drugs are most effective for killing cancer cells that are actively dividing when chemotherapeutic treatment is applied. Conversely, such drugs are relatively ineffective for the treatment of slow growing neoplasms. Carcinoma cells of the breast, lung and colorectal tissues, for example, typically double as slowly as once every 100 days. Id. at Table 301-1. Such slowly growing neoplasms present difficult chemotherapeutic targets.
Moreover, as with the emergence of resistant strains of pathogenic organisms, transformed cells can undergo further phenotypic changes that increase their resistance to chemotherapeutic drugs. Cancer cells can acquire resistance to genotoxic drugs through diminished uptake or other changes in drug metabolism, such as those that occur upon drug-induced gene amplification or expression of a cellular gene for multiple drug resistance (MDR). Id. at Ch. 301. Resistance to genotoxic drugs can also be acquired by activation or enhanced expression of enzymes in the cancer cell""s enzymatic DNA repair machinery. Therapies that employ combinations of drugs, or drugs and radiation, attempt to overcome these limitations. The pharmacokinetic profile of each chemotherapeutic drug in such a combinatorial regime, however, will differ. In particular, permeability of neoplastic tissue for each drug will be different. Thus, it can be difficult to achieve genotoxically effective concentrations of multiple chemotherapeutic drugs in target tissues.
Needs remain for drugs that can selectively destroy cells in a heterogenous cell population. Particular needs remain for drugs, including genotoxic drugs, that can selectively destroy cells of a pathogenic or undesired organism while preserving relatively unimpaired the viability of cells of a host or desired organism. Still more poignant needs remain for chemotherapeutic drugs, including genotoxic drugs, that can selectively destroy neoplastic or virally infected cells yet not significantly impair the viability of normal healthy cells in the body of an individual afflicted with cancer or a viral disease.
It is an object of this invention to provide a heterobifunctional compound that is genotoxic to selected cells in a heterogenous cell population. It is an object of this invention to provide a heterobifunctional compound that inflicts genomic lesions on selected cells in a heterogenous cell population. It is an object of this invention to provide a heterobifunctional compound that inflicts genomic lesions and impairs cellular repair of said lesions in selected cells in a heterogenous cell population. It is an object of this invention to provide a genotoxic agent or drug that can be xe2x80x9cprogrammedxe2x80x9d to destroy selected cells that are phenotypically distinguishable from nonselected cells in a heterogenous cell population. Another object of this invention is to expand the range of chemotherapeutic drugs available for the treatment of infectious and neoplastic diseases. Yet another object of this invention is to expand the range of infectious and neoplastic diseases that are susceptible to chemotherapy. These and other objects, along with advantages and features of the invention disclosed herein, will be apparent from the description, drawings and claims that follow.
In one aspect, the invention features a cell membrane permeant heterobifunctional compound suitable for destroying selected cells in a heterogenous cell population. The selected cells possess a cell component, such as a protein, that is absent or is present at significantly diminished levels in other, nonselected cells of the heterogenous cell population. Preferably, the cell component is intracellular. Most preferably, it is located within the cell nucleus or is naturally translocated to the nucleus from another intracellular site. In preferred embodiments, the cell component is a diffusible macromolecule having a molecular weight of at least about 25 kDa, more preferably at least about 40 kDa and still more preferably at least about 80 kDa. The present heterobifunctional compound is actively or passively transported across cell membranes, or diffuses through cell membranes. Thus, it can internalize within cells. It comprises a first agent that binds to cellular DNA to form a genomic lesion. The genomic lesion can be formed at a random or site-specific locus in cellular DNA. In certain embodiments, the first agent damages cellular DNA by forming one or more covalent bonds with nucleotide bases, the sugar-phosphate DNA backbone, or both. In other embodiments, the genomic lesion is formed by intercalation of the first agent into cellular DNA. Optionally, the first agent is a precursor that is converted into a DNA-reactive intermediate spontaneously or by exposure to physiological conditions, a cellular or secreted enzyme, product or byproduct of cellular metabolism, ionizing or nonionizing radiation, light energy, or the like. The genomic lesion so formed by interaction of the first agent with cellular DNA is potentially repairable by the cell""s enzymatic DNA repair machinery.
The first agent is linked to a second agent that binds to the cell component that is preferentially present in selected cells of the population. In some embodiments, the first and second agents are linked by a covalent bond. In other embodiments, the first and second agents are linked indirectly by covalent bonds to an organic linker. In still other embodiments, the first and second agents are linked by noncovalent interactions, such as electrostatic or hydrophobic interactions. Thus, in certain embodiments, the first and second agents become linked upon or following binding of the first agent to cellular DNA. The second agent forms a stable complex with the cell component. That is, the second agent interacts specifically with the cell component. Interaction can be noncovalent or covalent, and is energetically favored under intracellular, e.g., nuclear, conditions. As noted, the cell component is preferably a diffusible macromolecule, such as a protein. Alternatively, it can be a metabolite, ligand or cofactor that is specifically bound by a protein or another diffusible macromolecule present in the cell. In either circumstance, the complex comprises a macromolecular cell component found preferentially in the selected cells. The second agent thus localizes a sterically large cell component in the immediate vicinity of the genomic lesion. Preferably, the cell component is large enough to sterically obscure a segment of adjacent nucleosides extending from the lesion site for at least about five base pairs, more preferably at least about eight base pairs, still more preferably at least about twelve base pairs in both the 5xe2x80x2 and 3xe2x80x2 directions. As a result, the complex between the cell component and the second agent is effective for shielding or inhibiting repair of the genomic lesion formed by the binding of the first agent to cellular DNA. Formation of a sterically large complex at the lesion site hinders access by the cell""s enzymatic DNA repair machinery. As a result, shielded lesions persist in the genome and prejudice DNA replication, the expression of genes relevant to cell survival, and the like. Thus, the heterobifunctional compounds of the present invention are fatal to selected cells of the heterogenous cell population.
In certain embodiments, the second agent interacts specifically with a cell component that is relevant to the survival or proliferation of the selected cells. For example, the second agent can interact with a regulatory protein or enzyme involved in the control of cell proliferation. These include, but are not limited to, oncogene products (e.g., myc, ras, abl, and the like), tumor suppressor gene products (e.g., the nuclear phosphoprotein p53), and proteins that regulate initiation and progress through the cell cycle (e.g., cyclins and cyclin-dependent kinases). Alternatively, the second agent can interact with a transcription factor that controls or modulates the expression of one or more genes that are relevant to metabolic or secretory processes carried out by the selected cell. One such transcription factor is upstream binding factor (UBF), which controls the expression of ribosomal RNA genes and thus is pivotal to the function of the cell""s protein synthesis machinery. Second agents that specifically interact with transcription factors preferably mimic or resemble the natural genomic binding site for the particular transcription factor. That is, the transcription factor binds to the second agent with an affinity near (e.g., within about 100-fold) or preferably exceeding its affinity for the natural genomic binding site. Such second agents are referred to herein as xe2x80x9ctranscription factor decoysxe2x80x9d. Certain transcription factors, in addition to binding an endogenous genomic binding site, also bind to soluble ligands. Binding of these transcription factors to their cognate ligands modulates binding of the transcription factors to their endogenous genomic binding sites. That is, ligand binding confers or abrogates ability of the transcription factor to bind its cognate genomic site, or enhances or suppresses its ability to do so. Such transcription factors are accordingly referred to herein as ligand-responsive transcription factors. They have sometimes been referred to in the art as intracellular or nuclear receptors for soluble ligands. Second agents that recognize and bind to these transcription factors can mimic an activating or repressing ligand, such as estrogen or an estrogen analog or derivative. Heterobifunctional compounds comprising transcription factor decoys or ligand mimics thus are doubly fatal to the selected cell.
In another aspect, the present invention provides a method for the destruction of selected cells in a heterogenous cell population. The heterogenous cell population can comprise phenotypically distinguishable cells of a single phylogenetic species, or cells of two or more different phylogenetic species. The phylogenetic species can be unicellular or multicellular. The population can comprise cells in culture, cells withdrawn from a multicellular organism (e.g., a blood sample or tissue biopsy), or cells present in tissue or organs of a multicellular organism. It should be understood that the term xe2x80x9cmulticellular organismxe2x80x9d embraces mammals, including humans. The heterogenous cell population can comprise cells of both normal and transformed phenotypes. Thus, the population can comprise neoplastic or malignant cells. In the present method, selected cells of the heterogenous population are killed. xe2x80x9cSelected cellsxe2x80x9d are phenotypically distinguishable from other, nonselected cells in the heterogenous population in that they possess a cell component that is absent or is present at significantly diminished levels in nonselected cells. For example, the cell component is made or accumulates in the selected cells to levels that are about 5-fold in excess of the levels of the same or a similar cell component in nonselected cells. Preferably, the selected cells possess about a 10-fold excess of the cell component. More preferably, the selected cells possess about a 100-fold or higher excess of the cell component. In certain embodiments, the cell component is the expression product of a cellular or viral oncogene. In certain other embodiments, the cell component is the expression product of a mutant tumor suppressor gene. In still other embodiments, the cell component is a regulatory or enzymatic element of a nuclear protein complex that controls initiation of or progress through the cell cycle.
The present method involves contacting the heterogenous cell population with the cell membrane permeant heterobifunctional compound described herein. The population is incubated with the compound for a period of time sufficient for the compound to cross cell membranes and internalize within cells, including the selected cells. The first agent of the compound binds to cellular DNA, inflicting a genomic lesion. As noted above, the genomic lesion is potentially repairable. In selected cells, the second agent of the compound binds to the cell component, forming a complex at the genomic lesion site that sterically hinders access to the lesion by the cell""s DNA repair machinery, thereby inhibiting repair or xe2x80x9cshieldingxe2x80x9d the lesion. As a result, genomic lesions persist in the selected cells and contribute to their demise. That is, the present compound is preferentially genotoxic to the selected cells. In contrast, lesions in nonselected cells do not form complexes at the site of the genomic lesion, or form complexes with much lower frequency than in selected cells. Lesions in nonselected cells are therefore predominantly unshielded and remain accessible to the cellular DNA repair machinery. As a result, genomic lesions in nonselected cells are repaired. Lesion repair contributes to the survival of the nonselected cells. That is, the present compounds are relatively less genotoxic to nonselected cells. It is understood herein that the present compounds also may be internalized selectively by selected cells, regardless of whether the intracellular complex indeed is formed at the genomic lesion site. Selective internalization is expected to arise from the influence of intracellular complex formation in selected cells on chemical equilibrium dynamics between extracellular and intracellular levels of the present genotoxic compounds. Thus, the compounds of the present invention can be used to enhance selectively the uptake of DNA damaging first agents by selected cells in a heterogenous cell population. This process further contributes to the demise of selected cells.
As a result of the present method, the heterogenous cell population becomes depleted of selected cells. Embodiments of the present method wherein the selected cell component that is sequestered at the lesion site is a transcription factor are referred to as xe2x80x9ctranscription factor hijackingxe2x80x9d. In such embodiments, hijacking or sequestration of the transcription factor by the second agent at sites other than the factor""s natural genomic binding site still further contributes to the death of selected cells, by inducing disarray in one or more of the cell""s metabolic or secretory functions.
An advantage of the invention described herein is that heterobifunctional compounds can be engineered that are selectively fatal (genotoxic) to a great phenotypic and phylogenetic variety of selected cells. The term, xe2x80x9cprogrammable genotoxic drugsxe2x80x9d thus aptly sums up the flexibility and adaptability of the inventive concept disclosed herein.